Abstrakt
The JAK2V617F mutational burden in granulocytes and BFU-Es was determined by quantitative allele-specific PCR (qAS-PCR).3 The amplification of the exon 4 (T108A) and 9 (L393V) was done using the HotStarTaq Master Mix Kit (Qiagen, Valencia, CA) and the purified products were sequenced at University of Utah, Core DNA Sequencing Facility. Aliquots of DNA-free RNA were reverse-transcribed with SuperScript VILO (Invitrogen) and cDNA was amplified spanning the exon 4 - 16 and cloned into the pCR2.1-TOPO vector (TOPO TA Cloning kit; Invitrogen).
Single colonies positive for the recombinant plasmid were picked after overnight incubation, plasmid DNA was purified using the QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA) and the PCR insert was sequenced at University of Utah, Core DNA Sequencing Facility. JAK2 46/1 GGCC haplotype was determined on FastPCR 7500 instrument (Applied Biosystems) using Universal PCR Master Mix and TaqMan SNP assay on demand (rs3780367, C_27515396_10; rs10974944, C_31941696_10; rs12343867, C_319416 89_10), all samples were investigated in triplicate.